PROJECT SUMMARY/ABSTRACT Amphetamine-based drugs act on transport proteins that regulate mood and behavior, such as the dopamine and serotonin transporters, and are harnessed to therapeutically treat attention deficit hyperactivity disorder (ADHD). These drugs are also widely abused in the form of methamphetamine and 3,4- Methylenedioxymethamphetamine. Serotonin and dopamine transporters, widely targeted in the treatment of psychiatric disorders, belong to the neurotransmitter sodium symporter (NSS) family of secondary active transport proteins and are responsible for the reuptake of neurotransmitter from the synapse. Amphetamines, part of the larger monoamine releasing agents (MRA) drug family, act on serotonin and dopamine transporters to inhibit and reverse the reuptake process, leading to efflux of neurotransmitter into the synapse. Flooding of the synapse with neurotransmitter results in extreme stimulant effects and makes these drugs highly addictive. Efflux is thought to occur via two distinct modes, a reverse transport mode that follows the canonical alternating access model of transport, and a burst-efflux mode, characterized by large, transient fluxes of substrate. Burst efflux does not fit within the alternating access transport model. Therefore, two new models have been forwarded to explain its mechanism: a synchronous model where many transporters simultaneously release substrate in a synchronized fashion, or a channel-like model in which a single transporter operates via a channel-like mechanism to allow for large fluxes of substrate. We hypothesize that amphetamine-induced burst efflux is a channel-like mechanism of NSS?s that allows for large, transient rates of substrate translocation. To investigate this hypothesis, we have developed a single-molecule fluorescence resonance energy transfer (smFRET) assay that enables the quantification of transport processes at single transporter resolution. This assay capitalizes on the engineering of clamshell-like substrate binding proteins in combination with a novel approach for determining the orientation of single transporters. We will validate this new approach, for investigating transport, using the bacterial NSS homologue MhsT, which will enable us to characterize the distribution of rates and stochasticity of the transport process, intrinsic variables that cannot be addressed using ensemble-based methods. We will adapt this assay to the human serotonin transporter (hSERT) to determine if single transporters, of known orientation, exhibit transient, large efflux rates in the presence of amphetamines. We will characterize the rate, selectivity and directionality of these events to determine if they do, indeed, resemble a channel. This will then be leveraged to investigate the efflux properties of other MRAs to define correlations with specific drug phenotypes. We will use this new experimental paradigm to investigate amphetamine action on NSS?s, which may aid in the design of more effective, and less addictive, therapeutics. Additionally, the development of this platform will have a transformative effect on both the NSS field, as well as the wider secondary transport field, as it provides a completely novel modality of studying these proteins.